Tatsumi Daiki, Nanatani Kei, Koike Yuto, Kamagata Kiyoto, Takahashi Satoshi, Konno Ayumu, Furuta Tadaomi, Sakurai Minoru, Uozumi Nobuyuki
Department of Biomolecular Engineering, Graduate School of Engineering, Tohoku University, Aobayama 6-6-07, Sendai 980-8579, Japan.
Institute of Multidisciplinary Research for Advanced Materials, Tohoku University, Katahira 2-1-1, Sendai 980-8577, Japan.
Biochem J. 2017 May 30;474(12):1993-2007. doi: 10.1042/BCJ20161051.
ATP-binding cassette (ABC) transporters are ubiquitously present in prokaryotic and eukaryotic cells. Binding of ATP to the nucleotide-binding domains (NBDs) elicits major conformational changes of the transporters resulting in the transport of the substrate across the membrane. The availability of a crystal structure of the NBDs enabled us to elucidate the local structure and small-scale dynamics in the NBDs. Here, we labeled the ABC transporter MsbA, a homodimeric flippase from , with a fluorescent probe, Alexa532, within the NBDs. ATP application elicited collisional quenching, whereas no quenching was observed after the addition of ATP analogs or ATP hydrolysis inhibitors. The Alexa532-conjugated MsbA variants exhibited transition metal ion Förster resonance energy transfer (tmFRET) after the addition of Ni, and ATP decreased this Ni-mediated FRET of the NBDs. Structure modeling developed from crystallographic data and examination of tmFRET measurements of MsbA variants in the absence of ATP revealed the presence of metal ion-associated pockets (MiAPs) in the NBDs. Three histidines were predicted to participate in chelating Ni in the two possible MiAPs. Performing histidine-substitution experiments with the NBDs showed that the dissociation constant for Ni of MiAP2 was smaller than that of MiAP1. The structural allocation of the MiAPs was further supported by showing that the addition of Cu resulted in higher quenching than Ni Taken together, the present study showed that the NBDs contain two native binding sites for metal ions and ATP addition affects the Ni-binding activity of the MiAPs.
ATP结合盒（ABC）转运蛋白普遍存在于原核细胞和真核细胞中。ATP与核苷酸结合结构域（NBDs）的结合引发转运蛋白的主要构象变化，从而导致底物跨膜运输。NBDs晶体结构的可得性使我们能够阐明NBDs中的局部结构和小规模动力学。在这里，我们在NBDs内用荧光探针Alexa532标记了ABC转运蛋白MsbA，一种来自[具体来源未给出]的同型二聚体翻转酶。施加ATP会引发碰撞猝灭，而添加ATP类似物或ATP水解抑制剂后未观察到猝灭现象。添加Ni后，Alexa532偶联的MsbA变体表现出过渡金属离子福斯特共振能量转移（tmFRET），并且ATP降低了NBDs的这种Ni介导的FRET。根据晶体学数据开发的结构模型以及对无ATP情况下MsbA变体的tmFRET测量结果的检查揭示了NBDs中存在金属离子相关口袋（MiAPs）。预测三个组氨酸在两个可能的MiAPs中参与螯合Ni。对NBDs进行组氨酸取代实验表明，MiAP2对Ni的解离常数小于MiAP1。添加Cu比添加Ni导致更高的猝灭，这进一步支持了MiAPs的结构定位。综上所述，本研究表明NBDs包含两个天然的金属离子结合位点，并且添加ATP会影响MiAPs的Ni结合活性。
